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Photo-sensitive degron variants for tuning protein stability by light.

Usherenko S, Stibbe H, Muscò M, Essen LO, Kostina EA, Taxis C - BMC Syst Biol (2014)

Bottom Line: In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module.In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light.Two variants showed characteristics that are profoundly improved compared to the original construct.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology/Genetics, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, Marburg, 35043, Germany. taxis@staff.uni-marburg.de.

ABSTRACT

Background: Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus.

Results: We used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain.

Conclusions: In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well.

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Dark/light ratio of psd module variant abundance. The protein levels of psd module variants were measured in yeast cells grown in darkness or exposed to blue light (LED lamp, 465 nm, 30 μmol m-2 s-1) for 5.5 hours by fluorimeter measurements. Plasmid encoded constructs were used for the in vivo measurements; all variants were derived from the psd module construct PADH1-RFP-psd. At least six independent measurements were performed for each construct. Error bars: s.e.m.; mean values with a statistically significant difference (P<0.05) to the mean value of the wild type psd module are indicated by an asterisk.
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Fig3: Dark/light ratio of psd module variant abundance. The protein levels of psd module variants were measured in yeast cells grown in darkness or exposed to blue light (LED lamp, 465 nm, 30 μmol m-2 s-1) for 5.5 hours by fluorimeter measurements. Plasmid encoded constructs were used for the in vivo measurements; all variants were derived from the psd module construct PADH1-RFP-psd. At least six independent measurements were performed for each construct. Error bars: s.e.m.; mean values with a statistically significant difference (P<0.05) to the mean value of the wild type psd module are indicated by an asterisk.

Mentions: Next, the in vivo ratio of the abundance of the psd module variants in darkness and exposed to blue light was measured. We selected psd module variants with short half-life under blue light and at least moderate stability in darkness. The highest ratios were found in two mutants, K92R E132A E155G and K121M N128Y G138A. For both variants, the ratio was more than two times higher than in the wild type psd module. In the four variants K92R E132A N139E N148D E155G, K121M N128Y, G138A V142A R154G E155S, and N148E R154G E155S, we observed a moderate increase (about 1.4 fold) in the dark/light ratio (Figure 3, Table 1). Interestingly, all four variants with decreased stability at low illumination intensity (K92R E132A E155G, K121M N128Y, G138A V142A R154G E155S, and K121M N128Y G138A) show also an increase in dark/light ratio. The in vivo measurements highlighted the variants K92R E132A E155G and K121M N128Y G138A, which showed the highest dark/light switching factor and a faster degradation rate after blue-light illumination than the original construct (Figure 2, Figure 3, and Table 1). These two variants can be expected to be highly useful for in vivo manipulation of protein abundance by light.Figure 3


Photo-sensitive degron variants for tuning protein stability by light.

Usherenko S, Stibbe H, Muscò M, Essen LO, Kostina EA, Taxis C - BMC Syst Biol (2014)

Dark/light ratio of psd module variant abundance. The protein levels of psd module variants were measured in yeast cells grown in darkness or exposed to blue light (LED lamp, 465 nm, 30 μmol m-2 s-1) for 5.5 hours by fluorimeter measurements. Plasmid encoded constructs were used for the in vivo measurements; all variants were derived from the psd module construct PADH1-RFP-psd. At least six independent measurements were performed for each construct. Error bars: s.e.m.; mean values with a statistically significant difference (P<0.05) to the mean value of the wild type psd module are indicated by an asterisk.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4236813&req=5

Fig3: Dark/light ratio of psd module variant abundance. The protein levels of psd module variants were measured in yeast cells grown in darkness or exposed to blue light (LED lamp, 465 nm, 30 μmol m-2 s-1) for 5.5 hours by fluorimeter measurements. Plasmid encoded constructs were used for the in vivo measurements; all variants were derived from the psd module construct PADH1-RFP-psd. At least six independent measurements were performed for each construct. Error bars: s.e.m.; mean values with a statistically significant difference (P<0.05) to the mean value of the wild type psd module are indicated by an asterisk.
Mentions: Next, the in vivo ratio of the abundance of the psd module variants in darkness and exposed to blue light was measured. We selected psd module variants with short half-life under blue light and at least moderate stability in darkness. The highest ratios were found in two mutants, K92R E132A E155G and K121M N128Y G138A. For both variants, the ratio was more than two times higher than in the wild type psd module. In the four variants K92R E132A N139E N148D E155G, K121M N128Y, G138A V142A R154G E155S, and N148E R154G E155S, we observed a moderate increase (about 1.4 fold) in the dark/light ratio (Figure 3, Table 1). Interestingly, all four variants with decreased stability at low illumination intensity (K92R E132A E155G, K121M N128Y, G138A V142A R154G E155S, and K121M N128Y G138A) show also an increase in dark/light ratio. The in vivo measurements highlighted the variants K92R E132A E155G and K121M N128Y G138A, which showed the highest dark/light switching factor and a faster degradation rate after blue-light illumination than the original construct (Figure 2, Figure 3, and Table 1). These two variants can be expected to be highly useful for in vivo manipulation of protein abundance by light.Figure 3

Bottom Line: In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module.In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light.Two variants showed characteristics that are profoundly improved compared to the original construct.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology/Genetics, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, Marburg, 35043, Germany. taxis@staff.uni-marburg.de.

ABSTRACT

Background: Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus.

Results: We used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain.

Conclusions: In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well.

Show MeSH